EP2526785B1 - Procédé de production d'un phytoextrait à partir d'eaux agricoles et marc d'huile d'olive - Google Patents

Procédé de production d'un phytoextrait à partir d'eaux agricoles et marc d'huile d'olive Download PDF

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EP2526785B1
EP2526785B1 EP12169465.7A EP12169465A EP2526785B1 EP 2526785 B1 EP2526785 B1 EP 2526785B1 EP 12169465 A EP12169465 A EP 12169465A EP 2526785 B1 EP2526785 B1 EP 2526785B1
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pomaces
acid
range
filtration
treatment
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EP2526785A1 (fr
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Stefano GERMANI
Massimo Vitagliano
Daniele Pizzichini
Massimo Pizzichini
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PHENOFARM Srl
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
    • A23L19/03—Products from fruits or vegetables; Preparation or treatment thereof consisting of whole pieces or fragments without mashing the original pieces
    • A23L19/07—Fruit waste products, e.g. from citrus peel or seeds
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/63—Oleaceae (Olive family), e.g. jasmine, lilac or ash tree
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F9/00—Multistage treatment of water, waste water or sewage
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F1/00—Treatment of water, waste water, or sewage
    • C02F1/02—Treatment of water, waste water, or sewage by heating
    • C02F1/04—Treatment of water, waste water, or sewage by heating by distillation or evaporation
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F1/00—Treatment of water, waste water, or sewage
    • C02F1/24—Treatment of water, waste water, or sewage by flotation
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F1/00—Treatment of water, waste water, or sewage
    • C02F1/44—Treatment of water, waste water, or sewage by dialysis, osmosis or reverse osmosis
    • C02F1/444—Treatment of water, waste water, or sewage by dialysis, osmosis or reverse osmosis by ultrafiltration or microfiltration
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F1/00—Treatment of water, waste water, or sewage
    • C02F1/66—Treatment of water, waste water, or sewage by neutralisation; pH adjustment
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/30—Organic compounds
    • C02F2101/34—Organic compounds containing oxygen
    • C02F2101/345—Phenols
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
    • C02F2103/32—Nature of the water, waste water, sewage or sludge to be treated from the food or foodstuff industry, e.g. brewery waste waters
    • C02F2103/322—Nature of the water, waste water, sewage or sludge to be treated from the food or foodstuff industry, e.g. brewery waste waters from vegetable oil production, e.g. olive oil production
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2209/00—Controlling or monitoring parameters in water treatment
    • C02F2209/001—Upstream control, i.e. monitoring for predictive control
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2209/00—Controlling or monitoring parameters in water treatment
    • C02F2209/02—Temperature
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2303/00—Specific treatment goals
    • C02F2303/10—Energy recovery
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2303/00—Specific treatment goals
    • C02F2303/20—Prevention of biofouling
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C02F3/342—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the enzymes used
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W10/00—Technologies for wastewater treatment
    • Y02W10/30—Wastewater or sewage treatment systems using renewable energies

Definitions

  • VW vegetation waters
  • pomaces coming from olive milling Said process is based on combining physical-chemical and enzymatic pre-treatment methods, membrane tangential filtration and vacuum evaporation.
  • the method allows extracting eco-sustainable and efficient of the involved active ingredients.
  • the final extract is characterised by the presence of a high count of polyphenols and it is applied in the food, cosmetic, phytotherapeutic industries.
  • Olive-growing represents a crucial production industry for the entire Mediterranean area and Italy in particular.
  • olive trees are distributed on a mainly hilly surface which totally represents about a fifth of the surface designated for olive trees worldwide.
  • this industry suffers the drawback of considerable generation of wastes which in particular comprise vegetable tissues (leaves and prunnings) and actual olive oil production residues (VW, wet and used pomaces).
  • the latter are characterised by a strong pollution load, hence require suitable procedures so as to be able to be disposed according to the environmental laws in force (regarding Italy, DL 574 dated 11/11/96).
  • the scientific and industrial community has multiplied efforts to provide a solution to the problem regarding disposal of olive oil wastewaters.
  • wastes and residues deriving from the olive oil production chain may allow generating financial resources starting from non-conventional raw materials (wet pomaces, VW, used seedless pomaces) derived from the commercialization of active ingredients in production industries such as the food, cosmetic and phytotherapeutical industry.
  • DPPH radical allows studying the radical reduction kinetics and also calculating the EC 50 , i.e. the concentration of extract that reduces 50% of the radical.
  • EC 50 i.e. the concentration of extract that reduces 50% of the radical.
  • hydroxytyrosol and oleuropein aglycone are potent antioxidant and cardioprotective agents. Oleuropein reveals a coronary-dilating, hypoglycemic and anticholesterolemic activity and, similarly to hydroxytyrosol, it delays oxidation of LDLs( Low Density Lipoprotein ).
  • Hydroxytyrosol has been shown to reduce the gene expression of iNOS and COX-2 cell lines, thus preventing the activation of the NF-KB transcription factors, key factor in atherosclerosis, STAT-1 ⁇ and IRF-1.
  • a prevention of the activation of NF-KB was also observed in an ex-vivo study of monocytes of healthy volunteers exposed to consumption of olive oil, suggesting a possible anti-inflammatory effect by the oil.
  • Verbascoside also revealed in vitro immunomodulatory activity, in particular it increases the chemotactic activity of neutrophils.
  • the very verbascoside revealed, on PC12 neuronal cell culture, to have a protection activity against neurotoxicity induced by the 1-methyl-4-phenylpyridine (MPP+) ion, an ion which, activating caspase-3, causes serious oxidative stress in the cells.
  • MPP+ 1-methyl-4-phenylpyridine
  • a further polyphenol subclass (more specifically, a flavonoid subclass) of a particular biological interest, specifically present in the pulps of pigmented olives, is the class of anthocyanoside compounds or anthocyanines consisting, in particular, of glycoside derivatives of cyanidine, the most abundant of them being cyanidine-3-O-rutinoside.
  • Anthocyanosides are pigments specifically present in the skin of grapes and small fruits, marketed as standardized extracts of berries or marc residues, and widely studied in view of their biological activity.
  • the term anthocyanidines referred to the class of the corresponding non-glycosylated compounds (cyanidine being one of the major representatives thereof), has been created to designate the substances responsible for the colour of flowers, and is relevant to a group of water-soluble pigments responsible for the colours red, pink, violet and blue of most flowers and fruits.
  • the drugs containing anthocyanidines were used in galenic preparations for the treatment of the symptomatology connected with capillary fragility. Such compounds also show a high antioxidant power and are capable of protecting cells from oxidative damages caused by free radicals.
  • US2002198415 (A1 ), US2008090000 (A1 ), US2010216874 (A1 ) describe, starting from olive oil production industry wastes, how to obtain polyphenol-based extracts, through acid treatment of the VW and a prolonged storage thereof up to 12 months at a pH comprised between 1 and 6 with the aim of determining the conversion of oleuropein into hydroxytyrosol. After said incubation, the initial oleuropein was converted by 75-90% into hydroxytyrosol.
  • WO2007/013032 describes a process for recovering a concentrate rich in hydroxytyrosol from residues of the olive oil production industry, particularly vegetation waters and pruning residues (leaves).
  • Said process provides for the use, after extracting using a solvent (water or alcohol), of extraction systems with supercritical fluids, nanofiltration or, alternatively, reverse osmosis for recovering hydroxytyrosol and minor polar compounds.
  • the product thus obtained is an hydroxytyrosol-based extract.
  • WO2005/123603 describes a separation process based on membrane technologies specifically aimed at recovering compounds of interest from the VW. In such process, to the various tangential membrane filtration separation operations, there was introduced an initial filtration aimed at maximising the commercial useful polyphenol (such as hydroxytyrosol) contents to the detriment of the possible oleuropein still present in the VW.
  • the pre-treatment consists in acidifying still fresh VW to a pH of about 3 - 4.5, followed by an enzymatic hydrolysis.
  • an enzymatic hydrolysis After separating the liquid product thus treated by centrifuge there follows a series of cascade tangential filtration operations, which comprises a microfiltration followed by nanofiltration and lastly by a reverse osmosis process, obtaining from the various retentates polyphenol fractions with different degree of purification and from the reverse osmosis permeate purified water that can be used for producing beverages.
  • WO2008/090460 describes a further example of a process for recovering the hydroxytyrosol component from the olive oil production industry residues, in which there are not used milling wastes alone but also a given amount of green olives with the aim of obtaining a product particularly enriched in hydroxytyrosol.
  • the text proposes a first acid hydrolysis treatment at a temperature greater than the reflux temperature for the initial material (pomaces and green olive pulps), followed by a clarification of the resulting product (for example by filtration), in turn followed by a treatment on an ion exchange chromatography resin.
  • the product adsorbed on such column, after elution, may be in turn supplied to a second chromatography column loaded with a non-ionic adsorbent resin.
  • WO2009/016482 describes a process for the treatment of vegetal matter, including the olive VW. The authors propose a process comprising acidification, two steps of enzymatic lysis, providing for a passage for the separation of the solids between the two enzymatic lysis steps, microfiltration and vacuum evaporation.
  • An object of the present invention is to provide an innovative process for treating milling wastes capable of obtaining extracts rich in high molecular weight biologically active components, these components being lost by applying the milling wastes treatment methods known in literature.
  • the process of the present invention applied to the VW and pomaces coming from olive milling of cultivars Leccino and Carboncella, leads to the obtainment of a product whose composition and functional characteristics constitute an integral part of the present invention.
  • the invention is best described by the process of claim 1 and product claim 12.
  • the present invention aims at defining the process for obtaining an extract rich in high molecular weight biologically active components (MW > 500 Da), starting from the VW and pomaces coming from olive milling.
  • the method proposed herein is based on combining the physical-chemical and enzymatic pre-treatment methods, of the membrane tangential filtration technology (microfiltration) and vacuum evaporation.
  • the present process provides for a hydraulic agitation step during the enzymatic process which allows, operating using vegetation waters, the formation of a solid surface layer, defined cap , which is removed subsequently mechanically. Actually, it is the separation of said cap, whose composition is described herein, that allows obtaining the end product having the desired composition.
  • said process is applied to the VW and pomaces coming from olive milling of the cultivars Leccino and Carboncella.
  • the extract thus obtained was characterised from a composition and functional point of view and the properties thereof for specific applications in the food, cosmetic, phytotherapeutic industry are claimed herein.
  • the process consists in adopting complementary technologies (recirculating enzymatic reactor, tangential filtration, vacuum evaporation) for the extraction and the concentration of polyphenols contained in the matrices of interest (VW and pomaces).
  • Said process comprises: 1) collecting vegetation waters and pomaces after the olive milling process; 2) physical-chemical-enzymatic pre-treatment; 3) tangential filtration, preferably ceramic microfiltration (MF); 4) vacuum evaporation with reduction of the liquid extract obtained in step 3) into semisolid paste.
  • a scheme of said process is indicated in Figure 1 .
  • hydraulic agitation is used to indicate the agitation of the liquid mass obtained by operating a process for the recirculation of the very liquid eccentrically with respect to the walls of the reactor.
  • a scheme of said recirculation is indicated in figure 6 .
  • An opening arranged on the lower base of the reactor allows the exit of the liquid from the reactor introducing it into a conduit.
  • Said conduit returns into the reactor through an opening arranged on the upper base and descends again along the reactor up to re-introducing the liquid on the bottom of said reactor, in an eccentric position with respect to the walls of the reactor.
  • the liquid may circulate in said conduit due to a suitable pumping system, preferably selected from among a centrifuge pump and peristaltic pump, indicated by an arrow in figure 6 .
  • the product that is obtained through the process of the present invention is a dry or soft extract containing the entire polyphenolic content in the initial matrices, characterised by a total polyphenol content comprised between 40 and 100 ppm. Said product is intended for the food, cosmetic and pharmaceutical industries.
  • Said process comprises the following steps:
  • Said pre-treatment 1) is carried out both on the vegetable waters and on the pomaces with the aim of i) reducing and separating part of the load into solid suspensions present in the matrix (cellulosic fibres, oil globules, pulp residues), improving the filterability of the raw extract; ii) solubilising the polyphenolic and anthocyanic components which, remaining bound to the cellular wall residues, are difficult to recover.
  • the pomaces and the vegetable waters are characterised by a high content in solids which have a negative impact on the tangential filtration process, reducing the productivity values observed in the MF step by about 50%.
  • the cellulosic matrix entraps the polyphenols preventing the passage thereof into the solution and determining the lack of expression of the antioxidant action thereof.
  • the vegetable waters and the pomaces are treated immediately after terminating the production thereof in the miller, within 24 hours from the production thereof, preferably within 12 hours, so as to reduce the oxidation phenomena by the biophenols.
  • the vegetable waters and the pomaces produced are collected separately in the storage tanks and they are pre-treated separately by using the combination of pectolytic and cellulolytic enzymes, depolymerising enzymes which increase the release of the compounds of interest from the complex structure, typical of the olive cellular walls, in which they are enclosed.
  • This step provides for an acidification process for inhibiting the activities of the oxidoreductase such as polyphenol oxidase (PPO) and peroxidase (POD), both facilitated by the exposure of the air solution.
  • PPO polyphenol oxidase
  • POD peroxidase
  • the pre-treatment process allows reducing the suspended solids comprised between 40/60% and an increase of the total polyphenolic content (espresse in Gallic Acid Equivalents, GAE) comprised between 20/30%.
  • the pre-treatment allows recovering about 70-80% of the polyphenols present in the initial matrix.
  • the panel derived therefrom (used pomaces) due to the composition thereof in a nitrogen subsistence may constitute a valid organic soil improver for use in agriculture.
  • said process 2) for the pre-treatment of the vegetable waters and the pomaces comprises:
  • step a) acidification occurs by adding an acid, preferably selected from among citric acid, sulphuric acid, hydrochloric acid or mixture thereof.
  • said enzymes are food-grades and they are preferably produced from strains selected from among Aspergillus niger and Trichoderma longibrachiatum and they are added at amounts comprised between 0.02 and lo 0.1% weight/weight.
  • said hydraulic agitation is maintained over a period of time comprised between 2 and 6 hrs, preferably over about 4h at a T comprised between 30-50°C; in case of operation using pomaces, said hydraulic agitation is maintained for about 12-24 hrs at a T comprised between 50-80°C.
  • the hydraulic agitation and temperature conditions used in the pre-treatment lead to a progressive inclusion of air and oil particles in the solid phase, determining the floating of said solid phase over the liquid part, with a spontaneous separation of the solids.
  • the solid phase, the cap which is accumulated on the top part of the reaction mass also collects the oil inclusions that remain in the reaction mass, thus leading to a water/oil phase separation which contributes to the observed spontaneous floating.
  • Said cap is mainly constituted by colloidal substances, fats, vegetable fibres, salts, sugars and traces of polyphenol (0.5 - 1 mg/g total polyphenol expressed in GAE) and it constitutes 2-7% in weight with respect to the total mass of the vegetable water.
  • Said cap has a structure rich in air inclusions which increase the thickness thereof reducing the apparent density thereof, which reaches up to being comprised between about 0.7 and about 0.84 g/cm 3 .
  • the surface tends to cool down and become heavy, the air inclusions are reduced and thus the floating mass is compressed and the apparent density once again nears that of the vegetation waters (1.00 g/cm 3 ). Due to this reason, should the hydraulic agitation process be prolonged beyond 6 hours when working with VW or beyond 24 hours when working with pomaces, the mass tends to be re-compacted depositing on the bottom. It is thus essential for the present process the removal of said cap from the surface of the reaction mass during the thickening step, preferably after about 4 hrs of hydraulic agitation when working with VW and after about 24 hrs when working with pomaces.
  • Said step c) is carried out by pumping and sending to the subsequent filtering section the liquid mass which is collected on the bottom of the pre-treatment tank.
  • the product deriving from the pre-treatment step both for the pomaces and for the vegetable waters is constituted by a liquid (extract) characterised as shown in table 1: Table 1 Parameter Values measured in the liquid mass after the pre-treatment step pH 3-5,0 Electrical conductivity (mS/cm) 10-20 Water 86-94% Salinity (g/L) 8-12 P 2 O 5 1-2,2 K 2 O 5-8 FeO, MgO About 0,6 Dry matter (105°C) 6-14% Organic acids (g/L) 5-12 BOD 5 * (g/L of O 2 ) 50-150 COD *(g/L of O 2 ) 80-180 Fats (g/L) 0,3-23 Sugars (g/L) 20-35 Total polyphenols(g/L) 3-24 * BOD: biological oxygen demand * COD: Chemical oxygen demand
  • the liquid mass obtained through pre-treatment is thus subjected to said step 3) of tangential filtration.
  • the liquid mass deriving from the pre-treatment step is subjected to a perpendicular filtration step on a bag filter (60 micron cut-off ) and the extract is sent to the microfiltration section.
  • Said microfiltration is carried out using ceramic membrane modules with molecular size preferably comprised in the range between 0.1-1.4 microns with active filtering surface comprised between 0.20 and 1 m 2 per single module.
  • the ceramic modules may have an internal structure constituted by 8 to 85 channels.
  • the extract is filtered at a T comprised between 15 and 60°C, preferably between about 45 and about 50°C at a pressure comprised between 3 and 10 bars, preferably between about 5 and about 6 bars and it leads to progressive concentration.
  • a pressure comprised between 3 and 10 bars, preferably between about 5 and about 6 bars and it leads to progressive concentration.
  • the flow of the permeate with respect to time and the membrane surface is comprised between 15 and 100 l/m 2 h, preferably between 19 and 90 l/m 2 h.
  • the cut-off variability and module configuration allow minimising the impact on the performance of the filtration deriving from the natural compositive oscillations of the incoming matrix (solid content and polyphenol content, factors related to the seasonability of the matrix) and simultaneously maximise productivity in terms of amount of permeate over time.
  • the selected operating temperature allows an optimal permeation of the organic substance and it leads to a final product enriched in terms of the polyphenolic substances.
  • Said temperature is maintained under control through a refrigerating unit.
  • the fraction of concentrate comprises all the corpuscular particles (cellulosic fibres, oil globules, bacteria and vegetable cells) while the permeate is constituted by a solution, intense typically red-coloured due to the presence of antocyanic pigments having molecular weight comprised between 400 and 500 Da, and it comprises the entire pool of polyphenols and organic and inorganic dissolved substances (sugar, proteins, salts).
  • the microfiltration stage is carried out up to the obtainment of a concentration factor comprised between 5 and 20, upon reaching the preset VCR the process continuous through a dia-filtration process (DF).
  • the DF comprises i) adding to the volume of obtained concentrate with equivalent volumes of osmotic water; ii) new filtration up to the permeation of the entire volume of added water.
  • the DF allows increasing polyphenolic molecular content in the permeating fraction.
  • the added water volume equivalent to the concentrate obtained on the preset VCR is defined "diavolume”.
  • the permeate is sent to the vacuum evaporation unit.
  • the modules may be subjected to clogging, thus a procedure for controlling and containing this phenomenon, referred to as backpulsing, is carried out.
  • This procedure provides for a defined flow regime of the countercurrent permeate, with the aim of removing the deposit that is progressively formed on the membrane surface.
  • the backpulsing regime allows maintaining the productivity within acceptable levels and reducing the washing procedures and the concentration of detergent solutions.
  • the permeate is placed in a concentrator/evaporator operating with the following parameters: T comprised between 30 and 40°C, preferably about 35°C, distilled flow rate: 5-50 l/h, compressor delivery pressure comprised between 15 and 35 bars, preferably about 20.5-21.5 bars, compressor section pressure comprised between 4 and 9 bars, preferably about 5.6-5.9 bars, vacuum: 90-95 mbars, preferably 92 mbars, VCR: 5-20.
  • the evaporation goes on up to obtaining an end product with humidity comprised between 10 and the 30%, preferably between about 20 and about 30%.
  • the final product is obtained without requiring technological supports such as maltodextrins, gum arabic or others usually used in phyto-extracts but with the sole use of mechanical means and at low temperature.
  • step 4 may be followed by an atomization step to obtain a dry extract having humidity content comprised between 3 and 6%.
  • Phenolea Complex The compounds identified in the Phenolea Complex extract comprise phenolic acids, phenylpropanoids such as verbascoside and the derivatives thereof and flavonoid compounds, mainly luteolin and apigenin glycosides, but also quercetin and chrysoeriol as well as hydroxytyrosol.
  • Table 2 protein 2.00-3.00 g/100 g fats 0. 05-0.15 g/100 g dietary fibres 1.5-2.5 g/100 g ashes 5.5-6.5 g/100 g carbohydrates 59.00-63.00 g/100 g sugars 11.00-13.00 g/100 g sodium 340-380 mg/kg heavy metals: ⁇ 0.1 mg/kg phytopharmaceutical products Absent moulds ⁇ 10 UFC/1 g yeast ⁇ 10 UFC/1 g
  • Extracts obtained using the molecule claimed herein, more in particular, the extract referred to as Phenolea Complex revealed to be efficient for use in the preparation of nutraceutics, as antioxidant agents for balancing and reinforcing the immune system against damages caused by free radicals, as antioxidant agents for the protection of the DNA, protein and lipids against oxidation damage, as ingredients for functional foods and beverages intended for human and animal consumption.
  • Said extracts are applied in the food production industry, as antioxidants for preventing rancidification and as antimicrobial preservatives in fresh and/or frozen meat, sausage products, oven products, sauces and condiments generally in food products containing a fat part subjected to rancidification and/or in the food products subjected to bacterial proliferation.
  • the same can be used in the preparation of cosmetics or medicine for topical use with detoxifying and lenitive action for repairing age-related damages and/or damages caused by external or internal stress agents.
  • the pre-treatment step associated to the formation of the solid or semi-solid residue ( cap ) represents a crucial step of the process both in terms of optimisation of the performance of the production system and in terms of the product yield and quality.
  • the removal of said cap at the end of said step 2), without requiring using further pre-treatment steps, leads to:
  • the evaporation step allows obtaining a molass containing solely 10-30 % of water and a pool of polyphenols (simple and complex) such to facilitate the synergy between the different molecules and create the best conditions for the occurrence of the biological activity.
  • the 10-30% humidity content replicates the original chemical makeup present in the olive fruit.
  • Evaporation eliminating about 70-90% of water present in the loaded supply, concentrates the polyphenolic content up to obtaining a total polyphenol titre equivalent to 4-10 %.
  • said step allows a concentration of the liquid fraction without supplying heat, avoiding the heat stress of the end product and guaranteeing the preservation of the original pool of polyphenolic and anthocyanic molecules.
  • the absence of oxygen within the boiling boiler partly protects the product against biological contaminations.
  • the removal of water from the post-evaporated product facilitates a greater preservation capacity and a reduction of the biological contamination.
  • the product thus obtained maintains the water solubility of the present compounds unaltered facilitating the application thereof.
  • the end product is obtained without requiring generally used technological supports such as maltodexrins, gum arabic, silica, and thus avoiding phenomena related to the modification of the chemical substances present. Possible modifications include crystallisation of sugars, formation of lacquer, precipitation of salts, phenomena that could potentially jeopardise the functionality of the polyphenolic molecules of interest and interaction possibility thereof.
  • the addition of technological supports leads to an inherent disadvantage related to the addition of further solutes within the parent extract which would lead to lowering the titre of the substances of interest.
  • a further advantage lies in the possibility of reutilising the evaporated water in the treatment processes thus avoid the consumption of water from the water supply system or well.
  • the concentrator/evaporator operates under vacuum, optimising the yield of the energy applied.
  • distillate An amount of distillate is supplied to the microfiltration system for the dia-filtration technique. Focus has been made on the distillate produced, characterised by volatile substances initially present in the product introduced into a boiler which reflect the aroma of fresh olives and thus for the role that this fraction may play in the cosmetic and/or food industry.
  • Example 1 Pre-treatment of the VW, hydraulic agitation conditions - laboratory scale.
  • the disruption of the fibrous portion by the enzymes is at the base of the aggregation of the cap which does not occur in the absence of enzymes, unless in very small portions.
  • the distinctive characteristic of the three processes consists in the type of agitation carried out after the addition of the enzymatic preparation in each of the three samples.
  • Sample 1 was subjected to a central mechanical agitation, where a magnetic anchor was kept in agitation centrally on the bottom of the reaction mass at a regime of about 100 revolutions/minute.
  • Sample 2 was subjected to a side mechanical agitation, where a magnetic anchor was arranged at a decentred position on the bottom of the reaction mass and kept in agitation at a regime of about 100 revolutions/minute.
  • Sample 3 was subjected to hydraulic agitation, where a peristaltic pumping system operated a liquid recirculation in eccentric position with respect to the reaction mass, drawing the liquid from the lower base of the reaction mass and re-introducing the same, through a conduit, in a lower eccentric position with respect to said mass. Agitation was carried on for 120 minutes.
  • Figure 4 indicates the average weight of the moist cap as measured in each of the three samples.
  • Sample 1 revealed the formation of a vortex which did not allow the thickening of the cap.
  • Sample 2 revealed the formation of a calm area and a turbulence area, with the ensuing formation of an upper cap whose wet weight was about 40 g.
  • Sample 3 instead revealed a recovery of solids in the cap equivalent to at least 20% of the solids contained in said VW. In particular, the weight of the recovered cap is of about 90 g.
  • Figure 5 indicates an image of the cap as obtained in sample 2, subjected to lateral mechanical agitation, and in sample 3, subjected to hydraulic agitation.
  • the segment indicates the height of the obtained cap, equivalent to 0.8 cm in sample 2 and a 4.5 cm in sample 3.
  • Example 2 Pre-treatment of the VW, hydraulic agitation conditions - feasibility plant.
  • VW were introduced into a reactor and subjected to the conditions described in example 1: pH 3-3.5, 50°C.
  • the liquid mass was subjected to hydraulic agitation, as defined in the present invention.
  • the formation and the height of the cap was monitored at subsequent intervals. It was observed that the height peak of the cap was reached after 4 hrs of hydraulic agitation.
  • VW were subjected to a pre-treatment process as described in example 1, operating with hydraulic agitation.
  • the solid fraction of the cap was removed and the composition thereof was analysed.
  • the composition of said fraction is indicated in table 3
  • Table 3 Solid fraction "Cap” Centesimal composition % HUMIDITY 76.02 PROTEINS 4.73 FATS 9.84 DIETARY FIBRES 4.3 ASHES 1.02 SALTS, SUGARS AND OTHER ELEMENTS IN TRACES 5,1
  • Example 4 Microfiltration carried out on the liquid mass obtained from the pre-treatment of the VW and the pomaces.
  • Table 5 indicates the average polyphenolic profile observed in the VW of the Carboncella variety, before being subjected to extraction processes, as evaluated through HPLC/DAD Table 5: Vegetation waters Carboncella variety mg/L oleoside gluc deriv (390) 682.9 oleoside gluc (390) 2238.9 elenolic acid AE (242) 471.6 OH-Tyr glic (170) 55.3 OH-Tyr der. 71.9 OH-Tyr (hydroxytyrosol) 228.6 OH-Tyr gluc (316) 268.6 Tyr 74.3 Tyr gluc (300) 0.0 Tyr der.
  • Example 6 Extraction process starting from VW coming from cultivar Leccino and Carboncella and nutritional analysis of the obtained extract.
  • microfiltration was carried out using ceramic membrane modules with molecular size equivalent to about 0.14 microns with active filtering surface comprised between 0.20 and 1 m 2 per single module.
  • the extract is filtered at a T of 47°C at a pressure of 5 bars.
  • DF diafiltration
  • Phenolea Complex has the following chemical and nutritional characteristics.
  • Table 7 proteins 2.50 g/100 g fats 0.10 g/100 g dietary fibres 2.0 g/100 g ashes 6.00 g / 100 g carbohydrates 61.00 g/100 g sugars 12.00 g/100 g sodium 360 mg/kg heavy metals: ⁇ 0.1 mg/kg phytopharmaceutical products: Absent mould ⁇ 10 UFC/1 g yeast ⁇ 10 UFC/1 g total polyphenols (Expressed in Gallic Acid Equivalents) 45/100 mg/g
  • Example 7 Evaluation of the antioxidant properties of vegetable oils and animal fats of the Phenolea Complex extract compared with two commercial Rosemary extracts.
  • the Protection Factor of the Phenolea Complex extract is, in both concentrations used, exceeding the two types of commercial rosemary extracts used, in the same concentrations, in the comparison ( Figure 3a, 3b ).
  • Phenolea Complex extract reaches a stability index markedly greater than the two commercial Rosemary extracts.
  • the Rancimat method (Methrohm mod. 679) was used to determine the stability as a function of the oxidative decomposition.
  • Phenolea Complex The data indicated here reveals the considerable activity of the extract referred to as Phenolea Complex, which may thus be used, for example, in the sausage production industry, with the aim of protecting the product and especially replacing some additives (antioxidants).
  • Example 8 polyphenolic composition type of the Phenolea Complex extract.
  • the polyphenolic composition in terms of phenolic acids, phenolic alcohols, secoiridoids and flavonoids measured in the Phenolea Complex extract should be deemed equivalent - from a quality point of view - to the polyphenolic composition of the extra-virgin olive oil. Quantity-wise, the extract obtained through the process claimed herein has a greater polyphenolic concentration. Table 9 indicates the content in polyphenolic compounds identified in the Phenolea Complex extract with the mass spectrometry analysis LC/MS compared with the average compositions of a Sabina extra virgin olive oil.

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Claims (20)

  1. Procédé d'extraction et de concentration de composés polyphénoliques contenus dans des eaux de végétation et/ou dans des grignons obtenus par le traitement d'olives comprenant :
    1) la collecte du liquide consistant en eaux de végétation et/ou en grignons après le procédé de broyage des olives ;
    2) le prétraitement chimique, physique et enzymatique ;
    3) la filtration tangentielle à une température dans la plage de 45 à 50 °C, de préférence une microfiltration (MF) sur céramique, permettant ainsi d'obtenir une fraction de concentré et une fraction de perméat ;
    4) l'évaporation sous vide de la phase de perméat obtenue en 3) ;
    dans lequel ledit prétraitement 2) comprend :
    a) l'acidification à un pH entre 2,5 et 4 ;
    b) le traitement enzymatique par addition d'un pool d'enzymes cellulolytiques et pectolytiques, caractérisé en ce que pendant ledit traitement, le liquide est maintenu sous agitation hydraulique en exécutant un procédé pour la recirculation du même liquide de manière excentrique par rapport à la paroi du réacteur, dans lequel ladite agitation est maintenue pendant un temps de 2 à 6 H, de préférence pendant environ 4 H à une température dans la plage de 30 à 50 °C en cas de travail avec des eaux de végétation et ladite agitation hydraulique est maintenue pendant 12-24 H à une température dans la plage de 50 à 80 °C en cas de travail avec des grignons ;
    c) l'élimination du résidu solide ou semi-solide, appelé un chapeau, qui est collecté à la surface à la fin de ladite étape b) où ledit résidu a une densité comprise entre 0,7 et 0,85 g/cm3, dans lequel ladite élimination intervient en pompant et en envoyant la masse liquide collectée au fond de la cuve de prétraitement vers la section de filtration suivante et ledit chapeau constitue 2-7 % en poids par rapport à la masse totale de l'eau de végétation et contient environ 20 % des solides présents dans les eaux de végétation initiales.
  2. Procédé selon la revendication 1, dans lequel, après ladite étape 3) de filtration tangentielle, l'étape suivante est également mise en oeuvre :
    3') addition d'eau, de préférence d'eau osmotique, audit concentré, et diafiltration ultérieure jusqu'à la perméation de tout le volume d'eau ajouté, donnant ainsi une fraction de perméat qui est envoyée vers l'étape d'évaporation 4), avec la fraction de perméat obtenue en 3) ;
    dans laquelle ledit volume d'eau ajouté est de préférence égal au volume dudit concentré.
  3. Procédé selon la revendication 1, dans lequel ladite étape d'évaporation 4) se poursuit jusqu'à ce qu'un produit final ayant un taux d'humidité dans la plage de 10 à 30 %, de préférence de 20 à 30 %, soit obtenu.
  4. Procédé selon la revendication 1, dans lequel ladite étape d'évaporation 4) se poursuit jusqu'à ce qu'un produit ayant un taux d'humidité dans la plage de 50 à 80 % soit obtenu, et l'étape suivante est également mise en oeuvre :
    4') atomisation jusqu'à un produit final ayant un taux d'humidité dans la plage de 3 à 6 %.
  5. Procédé selon l'une quelconque des revendications 1 à 4, dans lequel lesdites eaux de végétation et/ou lesdits grignons sont collectés et traités dans les 24 heures suivant leur production, de préférence dans les 12 heures.
  6. Procédé selon la revendication 1, dans lequel ladite acidification intervient en ajoutant un acide, de préférence choisi parmi l'acide citrique, l'acide sulfurique, l'acide chlorhydrique ou des mélanges de ceux-ci.
  7. Procédé selon la revendication 1, dans lequel lesdites enzymes sont des enzymes de qualité alimentaire et sont de préférence produites par des souches choisies d'Aspergillus niger et Trichoderma longibrachiatum et sont ajoutées dans des quantités dans la plage de 0,02-0,1 % en pourcentage en masse.
  8. Procédé selon la revendication 1, dans lequel ledit résidu collecté à la surface à la fin de ladite étape b), le chapeau, consiste principalement en substances colloïdales, graisses, fibres végétales, sels, sucres et traces de polyphénols.
  9. Procédé selon la revendication 1, dans lequel ladite masse liquide provenant de l'étape de prétraitement 2) subit une étape de filtration perpendiculaire sur filtre à manche, de préférence avec un seuil de coupure d'environ 60 microns et est ensuite envoyée vers ladite section de filtration tangentielle.
  10. Procédé selon la revendication 1, dans lequel ladite filtration est mise en oeuvre avec des modules à membrane en céramique avec un seuil de coupure moléculaire allant de préférence de 0,1 à 1,4 micron ayant une surface de filtration active comprise entre 0,20 et 1 m2 par module individuel à une pression comprise entre 3 et 10 bar, de préférence d'environ 5 à environ 6 bar avec un flux de perméat dans le temps dans la plage de 15 à 100 l/m2H, de préférence de 19 à 90 l/m2H, dans lequel ladite filtration est poursuivie jusqu'à ce qu'un facteur de concentration dans la plage de 5 à 20 soit atteint.
  11. Procédé selon la revendication 1 ou 2, dans lequel dans ladite étape 4) d'évaporation sous vide le perméat obtenu dans lesdites étapes 3) et/ou 3') est placé dans un concentrateur/évaporateur fonctionnant avec les paramètres suivants : température dans la plage de 30 à 40 °C, de préférence d'environ 35 °C, débit de distillat : 5-50 l/H, pression de refoulement de compresseur dans la plage de 15 à 35 bar, de préférence d'environ 20,5-21,5 bar, pression d'aspiration de compresseur dans la plage de 4 à 9 bar, de préférence d'environ 5,6-5,9 bar, vide : 90-95 mbar, de préférence 92 mbar.
  12. Produit obtenu à partir d'eaux de végétation et/ou de grignons issus du traitement d'olives selon le procédé revendiqué dans l'une quelconque des revendications 1 à 11, dans lequel lesdites eaux de végétation et/ou lesdits grignons sont exposés à un prétraitement déterminant le flottement d'une phase solide sur la partie liquide avec une séparation spontanée des solides, dans lequel ladite phase solide est ensuite éliminée en pompant et en envoyant la masse liquide collectée au fond de la cuve de prétraitement vers la section de filtration suivante et ladite phase solide contient environ 20 % des solides présents dans les eaux de végétation initiales, dans lequel ladite filtration est une filtration tangentielle mise en oeuvre à une température dans la plage de 45 à 50 °C, de préférence une microfiltration (MF) sur céramique.
  13. Produit selon la revendication 12, dans lequel lesdites eaux de végétation et/ou lesdits grignons proviennent de l'opération de broyage d'olives des cultivars Leccino et Carboncella.
  14. Produit selon la revendication 13 qui comprend une quantité de fibres alimentaires entre 1,5 et 2,5 g/100 g.
  15. Produit selon la revendication 13, appelé Phenolea Complex, caractérisé par le profil chimique nutritionnel indiqué dans le tableau : protéines 2,00-3,00 g/100 g graisses 0,05-0,15 g/100 g fibres alimentaires 1,5-2,5 g/100 g cendres 5,5-6,5 g/100 g glucides 59,00-63,00 g/100 g sucres 11,00-13,00 g/100 g sodium 340-380 mg/kg métaux lourds : <0,1 mg/kg Produits phytopharmaceutiques : Aucun moisissures <10 UFC/1g levures <10 UFC/1g
  16. Produit selon la revendication 15, caractérisé par le profil de HPLC suivant : DÉTERMINATION DE BIOPHÉNOLS PAR HPLC Phenolea Complex BIOPHÉNOLS TOTAUX 280 nm mg/kg 45261 PROFIL DE PHÉNOLS NATURELS PHÉNOLS NATURELS TOTAUX 280 nm mg/kg 39257 ALCOOLS AROMATIQUES TOTAUX 280 nm mg/kg 21328 Hydroxytyrosol 280 nm mg/kg 20131 Tyrosol 280 nm mg/kg 1197 DÉRIVÉS D'OLEUROPÉINE 280 nm mg/kg 23005 DÉRIVÉS DE LIGSTROSIDE 280 nm mg/kg 1710 VERBASCOSIDE 280 nm mg/kg 1089 OLÉOCANTAL 280 nm mg/kg 1020 LIGNANES TOTAUX (Pinorésinol et Acétoxypinorésinol) 280 nm mg/kg 36 ACIDES PHÉNOLIQUES TOTAUX Acide protocatéchique, acide vanillique, acide caféique, acide p-coumarique, acide férulique 280 nm mg/kg 4784 FLAVONOÏDES TOTAUX 280 nm mg/kg 222 Lutéoline 280 nm mg/kg 222 Apigénine 280 nm mg/kg n.d. ACIDES SECOIRIDOÏDES TOTAUX 240 nm mg/kg 7695 Acide décarboxyméthyl élénolique 240 nm mg/kg 2408 Acide élénolique 240 nm mg/kg 5287
  17. Produit selon la revendication 12 ou 13 destiné à être utilisé dans les secteurs de l'alimentation, des cosmétiques et de la phytothérapie.
  18. Produit selon la revendication 12 ou 13 destiné à être utilisé dans la préparation de produits nutraceutiques, tels que des antioxydants pour équilibrer et renforcer le système immunitaire contre les dégâts provoqués par les radicaux libres, tels que les antioxydants pour la protection de l'ADN, les protéines et les lipides contre les dégâts oxydatifs, tels que les ingrédients pour les boissons et aliments fonctionnels destinés à la consommation humaine ou animale.
  19. Produit selon la revendication 12 ou 13 destiné à être utilisé dans la technologie alimentaire, en tant qu'antioxydant pour prévenir la rancidité et en tant que conservateur antimicrobien, seul ou en combinaison avec d'autres additifs alimentaires choisis de préférence parmi l'acide ascorbique, l'ascorbate de sodium, dans les viandes fraîches et congelées, les saucisses de type salami, les produits de boulangerie, les sauces et assaisonnements, et dans les aliments contenant une partie de graisse sujette à la rancidité et/ou dans les aliments sujets à la prolifération bactérienne en général et à la détérioration des couleurs.
  20. Produit selon la revendication 12 ou 13 destiné à être utilisé dans la préparation de cosmétiques ou de médicaments pour un usage local ayant une action antioxydante, détoxifiante et apaisante visant à réparer les dégâts liés à l'âge et/ou les dégâts provoqués par des agents stressants externes ou internes.
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US11918007B2 (en) 2017-12-14 2024-03-05 Universidade Do Porto Foodstuff composition comprising a derivate of olive pomace
IT202000023827A1 (it) 2020-10-09 2022-04-09 Manni Oil Soc Agricola S R L Processo integrato per la produzione ecosostenibile di olio extravergine di oliva di alta qualita’ e sottoprodotti micronizzati in economia circolare.

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